h. pylori virulence factor caga increases intestinal cell ... · gastritis, peptic ulcer disease...

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INTRODUCTIONHelicobacter pylori is a pathogenic Gram-negative bacterium thatcolonizes over 50% of the world’s human population. Colonizationwith H. pylori is linked to numerous gastric disorders includinggastritis, peptic ulcer disease and gastric adenocarcinoma (Blaserand Atherton, 2004). Although gastric cancer occurs in fewer than1% of people colonized by H. pylori (Amieva and El-Omar, 2008),it is still the second-most common cause of cancer mortalityworldwide (Peek and Blaser, 2002), and more than 50% of gastricadenocarcinomas can be attributed to infection with H. pylori(Asghar and Parsonnet, 2001). Most people infected with H. pylori,however, do not develop gastric cancer and the molecularmechanisms underlying this disparity have yet to be fully elucidated.

Although there are many factors that appear to contribute to thecarcinogenicity of H. pylori, strains that translocate the CagAprotein into host cells are significantly more likely to cause gastriccancer than strains lacking this ability. CagA is one of 28 geneproducts encoded by the cag pathogenicity island (cag PAI), a 40-kb stretch of DNA shown to be present in most strains isolated

from patients with severe gastric pathology (Censini et al., 1996).During infection with H. pylori, CagA is translocated into host cellsvia a type IV secretion system (TFSS), where it interacts with amultitude of host cell proteins. These interactions have beenshown to affect signal transduction pathways, the cytoskeleton andcell junctions (Bourzac and Guillemin, 2005).

After translocation into host cells by the H. pylori TFSS, CagAcan be phosphorylated by Src family kinases on tyrosine residueswithin conserved Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs (Selbach etal., 2002; Stein et al., 2002). After phosphorylation, CagA has beenshown to induce morphological changes in cultured epithelial cellsthrough interaction with a variety of host-cell proteins such as SHP-2, Met, Csk, Grb2 and ZO-1 (Amieva et al., 2003; Churin et al.,2003; Higashi et al., 2002; Tsutsumi et al., 2003; Mimuro et al., 2002).In addition to its phosphorylation-dependent effects, CagA has alsobeen shown to interact in a phosphorylation-independent mannerwith pathways associated with proliferation and inflammation(Suzuki et al., 2009). Although it is not yet clear which of thesemyriad interactions are required for the development of gastriccancer in persons colonized by H. pylori, the ability of CagA tointeract with components of the canonical Wnt signaling pathwayprovides a potential link between the observed oncogenic effectsof CagA and a host signaling pathway frequently deregulated ingastrointestinal cancers (Franco et al., 2005).

In addition to its role in early embryogenesis, the canonical Wntsignaling pathway plays a crucial role in regulating the proliferationand homeostasis of gastrointestinal epithelia. In normal stomachand intestinal epithelia, Wnt signaling has been shown to beimportant for proliferation, stem cell maintenance, and tissuerenewal (Barker et al., 2010; Sato et al., 2011; Pinto et al., 2003;Ootani et al., 2009; Sato et al., 2004). On the other hand, activationof Wnt signaling has been shown to result in cancers of the stomach

Disease Models & Mechanisms 6, 802-810 (2013) doi:10.1242/dmm.011163

1Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA2Department of Microbiology, Oregon State University, Corvallis, OR 97330, USA*Present address: Department of Medicine, Hematology Division, StanfordUniversity School of Medicine, Stanford, CA 94305, USA‡Present address: Aquaculture/Fisheries Center, University of Arkansas-Pine Bluff,Pine Bluff, AR 71601, USA§Author for correspondence ([email protected])

Received 18 October 2012; Accepted 25 February 2013

© 2013. Published by The Company of Biologists LtdThis is an Open Access article distributed under the terms of the Creative Commons AttributionNon-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), whichpermits unrestricted non-commercial use, distribution and reproduction in any medium providedthat the original work is properly cited and all further distributions of the work or adaptation aresubject to the same Creative Commons License terms.

SUMMARY

Infection with Helicobacter pylori is a major risk factor for the development of gastric cancer, and infection with strains carrying the virulence factorCagA significantly increases this risk. To investigate the mechanisms by which CagA promotes carcinogenesis, we generated transgenic zebrafishexpressing CagA ubiquitously or in the anterior intestine. Transgenic zebrafish expressing either the wild-type or a phosphorylation-resistant formof CagA exhibited significantly increased rates of intestinal epithelial cell proliferation and showed significant upregulation of the Wnt target genescyclinD1, axin2 and the zebrafish c-myc ortholog myca. Coexpression of CagA with a loss-of-function allele encoding the β-catenin destructioncomplex protein Axin1 resulted in a further increase in intestinal proliferation. Coexpression of CagA with a null allele of the key β-catenin transcriptionalcofactor Tcf4 restored intestinal proliferation to wild-type levels. These results provide in vivo evidence of Wnt pathway activation by CagA downstreamof or in parallel to the β-catenin destruction complex and upstream of Tcf4. Long-term transgenic expression of wild-type CagA, but not thephosphorylation-resistant form, resulted in significant hyperplasia of the adult intestinal epithelium. We further utilized this model to demonstratethat oncogenic cooperation between CagA and a loss-of-function allele of p53 is sufficient to induce high rates of intestinal small cell carcinomaand adenocarcinoma, establishing the utility of our transgenic zebrafish model in the study of CagA-associated gastrointestinal cancers.

H. pylori virulence factor CagA increases intestinal cellproliferation by Wnt pathway activation in a transgeniczebrafish modelJames T. Neal1,*, Tracy S. Peterson2,‡, Michael L. Kent2 and Karen Guillemin1,§

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CagA transgenic zebrafish model RESEARCH ARTICLE

and colon (Oshima et al., 2006; Powell et al., 1992; Fearon andVogelstein, 1990). Wnt pathway activity is tightly controlled viaregulation of the primary Wnt effector protein, β-catenin. β-catenin complexes with E-cadherin to form adherens junctionsbetween epithelial cells, and in the absence of Wnt ligand, is alsobound by Axin/APC/Gsk3β in the so-called ‘β-catenin destructioncomplex’, where it is targeted for proteosomal degradation. Uponbinding of Wnt by the co-receptors Frizzled and LRP, Axin1 issequestered at the membrane, preventing assembly of the β-catenin destruction complex. This results in cytoplasmicaccumulation of β-catenin and subsequent translocation of β-catenin into the nucleus. Upon nuclear translocation, β-cateninbinds the essential transcriptional cofactor TCF and initiatestranscription of Wnt target genes, including axin, myc and cyclingenes.

Non-phosphorylated CagA has been previously shown to disruptthe β-catenin–E-cadherin complex in cultured epithelial cells,causing cytoplasmic and nuclear accumulation of β-catenin, andsubsequent activation of the Wnt pathway (El-Etr et al., 2004; Suzukiet al., 2005; Murata-Kamiya et al., 2007). Additionally, CagA hasbeen shown to increase signaling through β-catenin via activationof phosphatidylinositol 3-kinase and/or Akt (Suzuki et al., 2009).Although the mechanisms of interactions of CagA with the Wntpathway have yet to be fully elucidated, it is clear both that CagA

is capable of activating Wnt signaling through β-catenin and thatinappropriate activation of Wnt signaling is potentially oncogenic.Understanding the wide variety of host cell interactions requiredfor H. pylori-induced pathogenesis has necessitated the use ofanimal models, and to date numerous primate and rodent modelshave been developed (Wirth et al., 1998; Lee et al., 1997; Ohnishiet al., 2008; Solnick et al., 1999). Although previously unexploitedin the study of H. pylori pathogenesis, the teleost fish Danio rerio(zebrafish) has emerged as a model organism for the study of varioushuman diseases, including leukemia (Feng et al., 2010), melanoma(Ceol et al., 2011; White et al., 2011) and intestinal neoplasia(Haramis et al., 2006). In lieu of a stomach, zebrafish possess ananterior digestive compartment known as the intestinal bulb. Thezebrafish intestinal bulb epithelium is columnar and non-ciliatedlike that of the mammalian stomach and expresses sox2 and barx1(Muncan et al., 2007), two mammalian stomach markers(Tsukamoto et al., 2005; Tissier-Seta et al., 1995; Kim et al., 2005).Unlike the mammalian stomach, however, it lacks the chief andparietal cell types. Nonetheless, the zebrafish intestinal bulb hasbeen proposed to share a common ontogeny with the mammalianstomach and its renewal is regulated by similar molecular pathways,including the Notch and Wnt pathways (Crosnier et al., 2005;Cheesman et al., 2011). Finally, the rapid development of thezebrafish intestinal tract makes it an ideal model for the study ofgastrointestinal development and disease (Faro et al., 2009).

Here, we describe the development of a novel transgenic modelsystem that simplifies the complexity of H. pylori infection to studythe effects of a single bacterial protein, CagA, on host cell biologyin the zebrafish intestine. We report that proliferation in thezebrafish larval intestinal epithelium is increased by transgenicexpression of CagA and that this increase occurs independently ofCagA phosphorylation. We demonstrate that expression of CagAinduces cytoplasmic and nuclear accumulation of the Wnt effectorβ-catenin, as well as activation of known Wnt target genes. Thegenetic tractability of the zebrafish system allowed us to exploregenetic interactions between CagA and a number of host signalingpathways. We show that CagA causes proliferation of the zebrafishintestinal epithelium via activation of the canonical Wnt signalingpathway downstream of or in parallel to the β-catenin destructioncomplex and upstream of the β-catenin transcriptional cofactorTcf4. Additionally, we demonstrate that long-term expression ofwild-type CagA, but not the phosphorylation-resistant form, issufficient to induce pathologic intestinal hyperplasia in adults andthat oncogenic cooperation between the cagA transgene and a loss-of-function allele of p53 results in high rates of intestinaladenocarcinoma and small cell carcinoma.

RESULTSGeneration of CagA-expressing transgenic zebrafish.In order to generate cagA transgenic animals, we cloned the cagAgene from H. pylori strain G27. Strain G27 was originally isolatedfrom Grossetto Hospital (Tuscany, Italy), and has been usedextensively in research on the CagA virulence factor (Amieva et al.,2003; El-Etr et al., 2004; Guillemin et al., 2002; Segal et al., 1999).The cloned gene was then 3�-tagged with EGFP to facilitate in vivovisualization of CagA expression. To express CagA ubiquitously inzebrafish, the cagA/EGFP fusion construct was connecteddownstream of the 5.3 kb beta-actin (b-) (Higashijima et al., 1997)

TRANSLATIONAL IMPACT

Clinical issueInfection with the bacterium Helicobacter pylori represents a major globalhealth burden that has been implicated in a wide range of gastric disorders,from acute gastric inflammation to stomach cancer. H. pylori strains that arecapable of translocating the bacterial effector protein CagA into host epithelialcells are associated with the highest risk of gastric cancer development. CagA-induced pathogenesis is multifactorial, and in vitro studies have reporteddifferent effects in diverse cell lines. Furthermore, CagA-induced oncogenesisis strongly associated with variations in host genotype, highlighting the needfor a genetically tractable in vivo model that faithfully recapitulates themechanisms underlying carcinogenesis in humans.

ResultsIn this manuscript, the authors report the development of a novel transgeniczebrafish system for the investigation of the H. pylori virulence factor CagA.This system recapitulates the major hallmarks of CagA pathogenesis observedin cell culture and murine models, while providing distinct advantages overprevious models. Using the zebrafish model, the authors demonstrate that theearly effects of CagA on intestinal epithelial proliferation require the functionof the canonical Wnt signaling component Tcf4, a β-catenin cofactor, but notAxin1, a component of the β-catenin destruction complex. They further reportthat co-expression of CagA with a loss-of-function allele of the tumorsuppressor p53 results in high rates of neoplastic transformation, therebyproviding the first direct in vivo evidence for oncogenic cooperation betweenCagA and p53.

Implications and future directionsThe CagA transgenic zebrafish model described herein presents several keyadvantages over current in vivo models. First, the rapid development of thezebrafish digestive tract makes it an ideal system for the study of CagA-associated gastrointestinal disease. Second, the ease of transgenesis via Tol2transposition enables the rapid introduction of additional alleles, facilitatingstructure-function studies that are difficult to perform in other vertebratemodels. Finally, the microbiota of CagA transgenic zebrafish is readilymanipulated or ablated, paving the way for future CagA gnotobiotic studies.

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promoter (supplementary material Fig. S1A). To facilitate intestine-specific expression of the fusion construct, we connected cagA/EGFPdownstream of a 1.6-kb fragment of the zebrafish intestinal fatty acidbinding protein (i-) (Her et al., 2004) promoter (supplementarymaterial Fig. S1B). By 6 days post-fertilization (dpf) b-cagA/EGFPtransgenic zebrafish exhibited ubiquitous fluorescence, whereas i-cagA/EGFP transgenic larvae exhibited fluorescence in the distalesophagus and anterior intestine (Fig. 1A,B). The phosphorylationstate of CagA has been previously shown to have significant effectson the type and severity of CagA-induced pathologies, so in orderto determine the role of CagA phosphorylation in the intestinalepithelium, we fused the previously described phosphorylation-resistant cagAEPISA allele (Stein et al., 2002) (supplementary materialFig. S1C) to EGFP and connected it downstream of the b-actinpromoter (supplementary material Fig. S1D). b-cagAEPISA/EGFPtransgenics exhibited ubiquitous fluorescence and were indistinctfrom b-cagA/EGFP fish (Fig. 1C). Expression of cagA mRNAs wasverified in transgenic animals by RT-PCR (Fig. 1D), and analysis ofrelative intestinal cagA transcript level in the transgenic lines viaquantitative real-time PCR revealed significantly elevated expression

of the cagA transgene when driven by the b-actin promoter ratherthan the i-fabp promoter (Fig. 1E).

CagA expression causes overproliferation of the intestinalepitheliumTo determine the effects of CagA expression on the larval zebrafishintestine, we examined wild-type and CagA transgenic animals at6 dpf, by which time autonomous feeding has begun, and at 15 dpf,by which time intestinal folding is complete (Ng et al., 2005). CagA-expressing zebrafish larvae showed normal intestinal development(Fig. 2A,B) and were histologically indiscernible from wild-typeclutch-mates (Fig. 2C,D). In addition, the CagA-expressing larvaeexhibited no gross abnormalities in cell junctions, as assessed bystaining with a pan-cadherin antibody (supplementary material Fig.S2). We next sought to establish the effects of CagA on larvalintestinal proliferation, because CagA had been previously shownto increase epithelial cell proliferation in vitro and in vivo (Mimuroet al., 2002; Nagy et al., 2011). To determine the proliferation stateof CagA-expressing intestines, we analyzed animals at 6 and 15 dpfthat had been exposed to the nucleotide analog 5-ethynyl-2�-deoxyuridine (EdU) for ~10 hours and counted S-phase nuclei in30 serial sections of the intestinal bulb. Expression of CagA resultedin a significant increase in EdU-labeled cells in all transgenic linesat 6 and 15 dpf (Fig. 2E,F). To determine whether this increase inproliferation had an effect on the cell census, we quantified totalepithelial cell number in single hematoxylin and eosin (H&E)-stained sagittal sections along the length of the intestine. We didnot observe any significant difference in total cell counts betweenCagA transgenics and wild-type animals at 6 and 15 dpf (Fig. 2G,H),indicating that expression of CagA caused increased turnover ofintestinal epithelial cells. Increased intestinal cell turnover wouldrequire an increase in cell death; however, consistent with previousreports and due to the transient nature of extruded apoptotic cells(Crosnier et al., 2005), we observed very few TUNEL-positive cellsin the intestines of wild-type and CagA-expressing animals(Fig. 2I,J), with no significant difference observed between the twogroups. Finally, the intestinal epithelia of b-cagA animals did notdisplay an increased number of local neutrophils at 8 dpf, indicatinga lack of CagA-induced intestinal inflammation at this stage(supplementary material Fig. S3).

CagA expression activates the Wnt pathway downstream of the β-catenin destruction complexWe had previously shown that epithelial cell proliferation in thezebrafish intestine is regulated by the Wnt pathway (Cheesman etal., 2011). In addition, previous studies had shown that CagA caninduce cytoplasmic and nuclear accumulation of the Wnt effectorprotein β-catenin and can activate transcription of canonical Wnttarget genes (Franco et al., 2005; Suzuki et al., 2009; Nagy et al.,2011). Accordingly, we examined whether CagA expression wascapable of activating the Wnt signaling pathway in the zebrafishintestine at different developmental stages. We first utilizedquantitative real-time PCR to assess the relative expression levelsof known Wnt target genes in dissected adult intestines. Transcriptlevels of the Wnt target genes c-myc (myca) (He et al., 1998), axin2(Yan et al., 2001) and cyclinD1 (Tetsu and McCormick, 1999) weremodestly increased in all CagA-expressing lines relative to the wild-type strain (Fig. 3A-C). We next asked whether CagA was capable

Fig. 1. Development of CagA+ transgenic zebrafish. (A)Ubiquitous CagA-EGFP fusion protein expression driven by the b-actin promoter. (B)UbiquitousCagAEPISA-EGFP fusion protein expression driven by the b-actin promoter.(C)Intestinal CagA-EGFP fusion protein expression driven by the i-fabppromoter. (D)RT-PCR of dissected larval intestine showing expression of cagAand the housekeeping control gene RPL13 at 6 dpf. (E)Quantitative RT-PCR ofdissected adult intestines showing relative expression levels of cagA transcriptin transgenic lines at 1 year of age. Expression levels were normalized to SDHAand β-actin; bars indicate mean ± s.d. of biological triplicates. Scale bars: 500 μm.

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of inducing β-catenin accumulation in epithelial cells of the larvalintestine, indicating activation of the Wnt pathway. CagAexpression caused a significant increase in the number of intestinalepithelial cells with cytoplasmic and nuclear accumulation of β-catenin as compared with wild-type animals (Fig. 3D,E). The factthat EdU labeling was not usually coincident with cytoplasmic andnuclear accumulation of β-catenin is probably due to the fact that,whereas relocalization of β-catenin is a transient event, the EdU-

labeled cells had undergone S-phase any time during the 12-hourlabeling period.

In order to assess the significance of CagA-induced β-cateninaccumulation, we next compared the intestinal β-cateninaccumulation observed in CagA-expressing animals to that of aknown Wnt signaling mutant, axin1tm213. The axin1tm213

homozygotes exhibit deregulated Wnt signaling as a result of amissense mutation in the Gsk3β binding domain of Axin1, whichprevents assembly of the β-catenin destruction complex. Thesemutants die as a result of craniofacial defects, but are viable until8 dpf, allowing study of the juvenile intestine (Heisenberg et al.,2001; van de Water et al., 2001). As expected, we observed increasesover wild-type and CagA-expressing animals in both the numberof proliferating cells and the number of cells featuring cytoplasmicand/or nuclear accumulation of β-catenin in the intestinal epitheliaof axin1tm213/tm213 mutants, consistent with constitutively activatedWnt signaling (Fig. 3E).

We reasoned that if CagA were capable of activating Wntsignaling upstream of the β-catenin destruction complex, thenaxin1tm213 homozygotes should be refractory to CagA-inducedaccumulation of β-catenin and levels of β-catenin accumulation inb-cagA; axin1tm213/tm213 double mutants should resemble those ofaxin1tm213 homozygotes. Instead, when we generated b-cagA,axin1tm213/tm213 fish, we found that expression of CagA in axin1homozygous mutants resulted in a dramatic increase in cellproliferation and β-catenin accumulation (Fig. 3F). Taken together,these data indicate that CagA is capable of causing sustainedactivation of canonical Wnt signaling in the intestinal epitheliumand that it does so either downstream of, or in parallel to, the β-catenin destruction complex. Furthermore, CagA-inducedaccumulation of β-catenin was strongly correlated with increasedepithelial proliferation (Fig. 3G,H), suggesting that CagA stimulatesproliferation through activation of the Wnt pathway.

CagA-dependent overproliferation of the intestinal epitheliumrequires tcf4To determine whether CagA-induced overproliferation of theintestinal epithelium is dependent on canonical Wnt signalingdownstream of the β-catenin destruction complex, we utilized anull allele of the essential β-catenin transcriptional cofactor, Tcf4(Muncan et al., 2007). We reasoned that if the pro-proliferativeeffects of CagA were acting upstream of Tcf4, rates of intestinalproliferation in i-cagA; tcf4exl double mutants should be identicalto those observed in tcfnull animals. As previously observed, i-cagAanimals showed a significant increase in proliferation over the wildtype, whereas tcf4exl/exl mutants showed levels of intestinalproliferation similar to wild-type animals (Fig. 4). Rates of intestinalproliferation in i-cagA; tcf4exl/exl larvae were statisticallyindistinguishable from wild-type and tcf4 exl/exl mutants, indicatingthat CagA requires Tcf4 function to increase intestinal epithelialproliferation. This result places activation of the Wnt signalingpathway by CagA downstream of or in parallel to Axin1 andupstream of Tcf4 (supplementary material Fig. S4).

CagA expression causes phosphorylation-dependent intestinalhyperplasia in adult zebrafishH. pylori-associated gastric adenocarcinoma occurs as a result oflifelong exposure to the bacterium, with CagA+ strains posing a

Fig. 2. CagA expression causes overproliferation of the intestinalepithelium. (A,B)H&E stained sagittal sections of wild-type (A) and b-cagAtransgenic (B) zebrafish intestine at 6 dpf. (C,D)H&E stained sagittal sections ofwild-type (C) and b-cagA transgenic (D) zebrafish intestine at 15 dpf.(E,F)Intestinal epithelial cell proliferation at 6 dpf (E) and 15 dpf (F). Barsrepresent proliferation (mean ± s.e.m.) as a percentage of wild-type; n=10,*P<0.05 using one-way ANOVA with Tukey’s test. (G,H)Total intestinalepithelial cell counts of single H&E stained midline sagittal sections at 6 dpf (G)and 15 dpf (H). (I,J)TUNEL-positive cells in the intestinal epithelium at 6 dpf (I)and 15 dpf (J). Scale bars: 10 μm.

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significantly greater cancer risk (Asghar and Parsonnet, 2001). Inorder to study the long-term effects of CagA exposure in our model,we performed histological analysis of adult b-cagA, i-caga and b-cagaEPISA animals at 1 year of age. Wild-type adults (18 monthspost-fertilization) served as controls. Upon examination, nohyperplastic or neoplastic lesions were found in any of the wild-type controls (Fig. 5A,G; supplementary material Table S1). Aproportion of the b-cagA and i-cagA individuals exhibitedsignificant intestinal epithelial hyperplasia at 1 year of age(Fig. 5B,C). Surprisingly, despite the significant increases inproliferation and Wnt activation observed in younger b-cagaEPISA

animals, no hyperplasia was observed in age-matched adults of thisgenotype (Fig. 5D). These data suggest that although thephosphorylation-independent activation of Wnt signaling by CagAis sufficient to induce sustained overproliferation of the larvalintestinal epithelium, it is not sufficient to induce significanthyperplastic changes in the adult intestinal epithelium, as seen inthe groups expressing the non-mutant CagA, either ubiquitouslyor in an intestine-specific manner.

Coexpression of the cagA transgene with a p53 loss-of-functionallele results in high rates of intestinal adenocarcinomaThe tumor suppressor gene p53 is frequently mutated in diffuse-and intestinal-type gastric cancers (Nobili et al., 2011; Ranzani etal., 1995), and gastric adenocarcinomas isolated from CagA+ H.pylori-infected patients exhibit frequent mutation in p53 (Shibataet al., 2002). Additionally, CagA has been shown to subvert thetumor suppressor function of the apoptosis-stimulating proteinASPP2 in cultured cells, leading to enhanced degradation of p53(Buti et al., 2011). In order to examine the potential for oncogeniccooperation between the cagA transgene and p53 we bred b-cagAand i-cagA animals to animals homozygous for a loss-of-functionallele of p53 (tp53M214K) to obtain b-cagA; tp53M214K/M214K or i-cagA;tp53M214K/M214K animals. The zebrafish ortholog of p53 is highlyconserved in both structure and function and the tp53M214K DNA-binding domain mutation is orthologous to methionine 246missense mutations previously identified in human tumors (Storerand Zon, 2010). At 1 year post-fertilization, all of thetp53M214K/M214K fish failed to thrive and exhibited high rates ofocular malignant peripheral nerve sheath tumors, recapitulatingprevious studies using this p53 allele (Berghmans et al., 2005). Aninsufficient number of b-cagA; tp53M214K/M214K individuals survivedto this time point for analysis, but we were able to examine smallnumbers of both tp53M214K/M214K and i-cagA; tp53M214K/M214K

(Fig. 5E,F) lines. In both lines, we observed examples of intestinalepithelial hyperplasia and definitive neoplasia (Fig. 5G).

In the affected genotypes displaying hyperplastic changes, theintestinal mucosa was thrown into irregular and haphazard foldslined by a ragged and thickened epithelium often 2-6 cells deepwith pseudostratification of nuclei, which was most prominentwithin invaginations between the mucosal villi (mucosal sulci).Infolding of the hyperplastic epithelium frequently resulted information of mucosal pseudocrypts, with the most severely affectedintestines also displaying frequent epithelial fusion betweenadjacent mucosal folds. In addition, numerous aponecroticintestinal epithelial cells were observed and directly reflected rapidepithelial cell proliferation and turnover. Small numbers of achronic inflammatory cell infiltrate, composed mostly of

Fig. 3. CagA activates canonical Wnt signaling in the intestinalepithelium. (A-C)Quantitative RT-PCR data showing relative expression levelsof the Wnt target genes mycA (A), cyclinD1 (B) and axin2 (C). Expression levelswere assayed in dissected adult intestines and normalized to SDHA and β-actin; bars indicate mean ± s.d. of biological triplicates. (D-G)Immunofluorescence micrographs showing proliferating cells (EdU,green, 10-hour label) and cells with nuclear and cytoplasmic accumulation ofβ-catenin (red staining and white arrowheads) in intestinal cross-sections ofwild-type (D), b-cagA (E), axin1tm213 (F) and b-cagA; axin1tm213 (G) animals at 6dpf. (H)Quantification of proliferating (EdU+) cells. (I)Quantification of cellswith nuclear and cytoplasmic accumulation of β-catenin.

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lymphocytes and few eosinophilic granule cells, were seenpercolating through the hyperplastic epithelium in many areas. Fociof dysplastic intestinal epithelial cells were often identified inhyperplastic areas, usually within mucosal sulci. Dysplastic cellsdemonstrated progressive disorganization including ‘piling-up’ ofcells and loss of nuclear polarity, nuclear and cytologicpleomorphism, hyperchromatic elongated nuclei andinconspicuous nucleoli with sparse cytoplasm (increased nuclearto cytoplasm ratio) and occasional bizarre mitotic figures. In allcases where dysplastic cells were observed, there was no invasionthrough the basement membrane (i.e. carcinoma in situ) exceptfor one fish in the tp53M214K/M214K group, which had a solitarymaxillary (upper jaw) focus of carcinoma in situ within theoropharyngeal cavity. When definitive intestinal neoplasia was seen,adenocarcinoma was most often found in the anterior intestine andsmall cell carcinoma in the anterior or mid-intestine.

Adenocarcinomas displayed variable degrees of differentiation,ranging from well to poorly differentiated, with a tendency to formdisorganized and cribrose acinar-like pseudocrypts that penetrateddeep into the lamina propria, in the absence of an intercedingbasement membrane. Individual tumor cells had hyperchromatic,ovoid to elongated nuclei with granular chromatin, multiple smallnucleoli and sparse basophilic cytoplasm. In less differentiatedadenocarcinomas, bizarre mitotic figures were occasionally seen.Locally extensive fibrogenesis within the lamina propria(intraproprial desmoplasia) and variable numbers of chronicinflammatory cell infiltrates, comprised of intermingledlymphocytes and eosinophilic granule cells, were often associatedwith the adenocarcinomas. The two small cell carcinomas identifiedin the i-cagA; tp53M214K/M214K group were composed of denselycellular nests of polygonal to fusiform cells, lacking an organoidpattern, which infiltrated deep into the lamina propria and werenot associated with pseudocrypts. Individual neoplastic cells withinnests had pleomorphic, deeply basophilic nuclei with densegranular chromatin, inconspicuous nucleoli and minimalcytoplasm. Solitary necrotic tumor cells were seen in some of thenests, accompanied by small aggregates of lymphocytes.Lymphovascular invasion and distant metastasis was not observedin either of the tumor types. Incidence and overall severity of lesionswithin the expression domain of the cagA transgene were higherin i-cagA; tp53M214K/M214K animals than in the correspondinganatomical region of tp53M214K/M214K animals (Fig. 5G;supplementary material Table S1). These data indicate thatexpression of CagA with concomitant p53 loss is sufficient to inducehigh rates of adenocarcinoma and small cell carcinoma in thezebrafish intestine and demonstrate the utility of our model for thestudy of CagA-associated gastrointestinal cancers.

DISCUSSIONHere, we describe the development of a novel in vivo model of CagA-induced intestinal pathology in zebrafish that recapitulates majorhallmarks of CagA pathogenesis observed in cell culture and murinemodels such as increased epithelial proliferation, cellularaccumulation of β-catenin and intestinal hyperplasia (Ohnishi et al.,2008; Mimuro et al., 2002; El-Etr et al., 2004; Suzuki et al., 2005;Murata-Kamiya et al., 2007; Nagy et al., 2011). We utilized transgenicexpression of CagA to investigate how the H. pylori virulence factorCagA is able to disrupt normal programs of intestinal epithelial

renewal via activation of an important host signaling pathway, theWnt pathway, to cause significant overproliferation of an intactepithelium in vivo. We show that activation of canonical Wntsignaling upstream of the essential β-catenin cofactor Tcf4 anddownstream of the β-catenin destruction complex is required forthe early effects of CagA on intestinal epithelial proliferation.

We further utilized our novel transgenic zebrafish system todemonstrate that long-term expression of CagA is sufficient to causeintestinal hyperplasia in adult zebrafish. Notably, although expressionof the phosphorylation-resistant b-cagAEPISA allele is capable ofinducing significant sustained overproliferation of the larval intestinalepithelium coupled with increased Wnt activation, it failed to inducesignificant intestinal hyperplasia in adult animals. These datacorroborate a previous study using a CagA transgenic mouse model,which demonstrated that the ability of CagA to induce severeepithelial hyperplasia in vivo is correlated with its capacity to bephosphorylated by host kinases (Ohnishi et al., 2008). It is possiblethat the activation of Wnt signaling by CagA and subsequentinduction of proliferation act in concert with further oncogenicstimuli, which might occur in the form of previously observedphosphorylation-dependent events such as epithelial depolarization(Amieva et al., 2003) or ERK activation by CagA (Higashi et al., 2004).These data illustrate the utility of long-term in vivo modeling of CagApathogenesis because the cumulative effects of CagA expressioncannot be predicted from the transient cellular responses it elicits.

Host genetics play a significant role in the development of H.pylori-associated gastric cancer. For example, certain alleles of thehost genes p53, IL-1β and IL-10 are strongly correlated with thedevelopment of gastric adenocarcinoma in H. pylori-infectedhumans (Shibata et al., 2002; El-Omar et al., 2003). Transgenicexpression of CagA in mice was sufficient to cause gastric andintestinal carcinomas, but these only developed in less than 5% ofthe animals (Ohnishi et al., 2008). We observed high rates ofintestinal neoplasia in our CagA transgenic zebrafish model whenexpressed with a mutant allele of the tumor suppressor p53. Thesedata provide the first direct in vivo evidence for oncogeniccooperation between CagA and p53 and provide a robust modelof CagA-induced carcinoma. Our results are consistent withprevious findings of increased p53 mutational frequency in H.

Fig. 4. CagA-dependent overproliferation of the intestinal epitheliumrequires tcf4. Intestinal epithelial cell proliferation at 15 dpf. Bars representproliferation (mean ± s.e.m.) as a percentage of wild-type; n=10, *P<0.05 usingone-way ANOVA with Tukey’s test.

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pylori-associated gastric cancer cases (Shibata et al., 2002) andcorroborate a previous study establishing CagA as a bona-fideoncoprotein (Ohnishi et al., 2008). More importantly, these datasupport the use of our model in the screening of putative gastriccancer susceptibility loci for oncogenic cooperation with CagA.

MATERIALS AND METHODSEthicsAll zebrafish experiments were carried out in strict accordance withthe recommendations in the Guide for the Care and Use ofLaboratory Animals of the National Institutes of Health. TheUniversity of Oregon Animal Care Service is fully accredited bythe Association for Assessment and Accreditation of LaboratoryAnimal Care and complies with all United States Department of

Agriculture, Public Health Service, Oregon State and local areaanimal welfare regulations. All activities were approved by theUniversity of Oregon Institutional Animal Care and Use Committee(Animal Welfare Assurance number A-3009-01).

AnimalsTransgenic zebrafish were developed using the Tol2kit aspreviously described (Kwan et al., 2007). tp53M214K (Berghmanset al., 2005), and axin1tm213 (Heisenberg et al., 2001) animals wereobtained from Monte Westerfield (University of Oregon) andtcf4exI (Muncan et al., 2007) from Tatjana Piotrowski (Universityof Utah). All zebrafish experiments were performed usingprotocols approved by the University of Oregon Institutional Careand Use Committee, and following standard protocols(Westerfield, 2007). CagA transgenics can be obtained bycontacting the corresponding author.

EdU labeling and detectionZebrafish larvae were immersed in 100 μg/ml EdU (A10044;Invitrogen) with 0.5% DMSO for 8-12 hours, fixed overnight at 4°C(4% paraformaldehyde in PBS) with gentle shaking, processed forparaffin embedding and cut into 7-μM sections. Slides were thenprocessed using the Click-iT EdU Imaging Kit (C10337, Invitrogen).EdU-labeled nuclei within the intestinal epithelium were countedover 30 serial sections, beginning at the intestinal-esophageal junctionand proceeding caudally into the intestinal bulb.

TUNEL stainingStaining was carried out using the Click-iT TUNEL Imaging Assay(C10245, Invitrogen). TUNEL-positive cells within the intestinalepithelium were counted over 30 serial sections, beginning at theintestinal-esophageal junction and proceeding caudally into theintestinal bulb.

ImmunohistochemistryImmunohistochemistry was carried out on paraffin sections aspreviously described using anti-β-catenin (1:1000; C2206 rabbitpolyclonal, Sigma) (Cheesman et al., 2011).

Fig. 5. CagA expression causes phosphorylation-dependent intestinalepithelial hyperplasia and induces adenocarcinoma formation incombination with p53 loss. (A-F)H&E stained sagittal sections of adultzebrafish intestine (A) Wild-type intestine at 18 months post-fertilization (mpf)showing normal intestinal architecture, with a single layer of epithelial cellslining the mucosal folds. (B,D)b-cagA (B) and i-cagA (D) intestines at 12 mpf,displaying mucosal fold epithelial hyperplasia, dysplasia within mucosal sulciand mucosal fold fusion. (C)b-cagAEPISA intestine at 12 mpf showing normalintestinal architecture, identical to wild-type. (E)i-cagA; tp53M214K/M214K smallcell carcinoma with small nests of neoplastic cells in the lamina propria(arrowheads). Inset depicts higher magnification of tumor cells; the crossmarks the epithelium. (F)i-cagA; tp53M214K/M214K adenocarcinoma, poorlydifferentiated, invade into the lamina propria with complete disorganizationof the epithelium, as shown by goblet cells randomly scattered throughout(arrowheads). (G)Summary of intestinal histological abnormalities observed inadult CagA-expressing animals as a result of a blinded histological analysis ofH&E stained sections (wild-type, n=22; b-cagA, n=24; b-cagAEPISA, n=18; i-cagA, n=19; tp53M214K/M214K, n=5; i-cagA/tp53M214K/M214K, n=7). Scale bars: 25 μm.

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HistopathologyHistopathological analysis of H&E stained sections was performedby pathologists with expertise in laboratory fish (T.S.P. andM.L.K.) in a blinded manner. For each adult zebrafish genotype,four consecutive sagittal serial sections of the entire intestinaltract, anterior to posterior, were evaluated for epithelialhyperplasia, dysplasia and the presence of neoplasia. Classificationof intestinal epithelial hyperplasia included two or more of thefollowing criteria: epithelial cell nuclear pseudostratification,multi-layering of mucosal fold epithelial cells and formation ofpseudocrypts, which indicated extensive infolding of hyperplasticepithelium lining the intestinal mucosal folds. Dysplastic changesof the intestinal epithelial cells, observed in several fish withinthe hyperplastic intestinal epithelium, were classified as anincreased nuclear to cytoplasm ratio, nuclear hyperchromatismwith indiscernible nucleoli, ‘piling-up’ of epithelial cells, loss ofnuclear polarity (i.e. loss of basally oriented epithelial cell nuclei)and abnormal mitotic figures. Classification of intestinaladenocarcinoma included the following criteria: invasivecribriform pseudocrypts that interfaced directly with the laminapropria in the absence of an interceding basement membrane,disorganized histoarchitectural patterns of the pseudocrypts,loss of differentiation from well-defined pseudocrypts to completeabsence of acinar-like structures and a desmoplastic response tothe neoplastic cells. Small cell carcinoma was classified as denselycellular and discrete small sheets and nests of tumor cells withinthe lamina propria, with minimal cytoplasm, that lacked anorganoid growth pattern. Intratumoral inflammatory infiltrateswere also accounted for and classified by chronicity and cell type.Other proliferative lesions, which occurred in only one fish, aredescribed in the Results section.

Quantitative RT-PCRReference gene testing was performed using the geNorm referencegene selection kit (Primerdesign) and qBasePLUS software (Biogazelle).Baseline, threshold and efficiency calculations were performed usingLinRegPCR software (Ruijter et al., 2009). Quantitative RT-PCRreactions were performed using the SYBR FAST qPCR kit (KapaBiosystems) on a StepOnePlus Real-Time PCR System (AppliedBiosystems) using primers listed in supplementary material TableS2. Expression data were normalized to the geometric mean of thereference genes using StepOne (ABI) software.

Myeloperoxidase stainingMyeloperoxidase (Mpo) staining was carried out using the LeukocytePeroxidase (Myeloperoxidase) Staining Kit (Sigma-Aldrich). Mpo-positive cells within the intestinal epithelium were counted over 30serial sections, beginning at the intestinal-esophageal junction andproceeding caudally into the intestinal bulb.

Statistical analysisAll statistical analyses were performed with Graph-Pad Prismsoftware.ACKNOWLEDGEMENTSWe thank Erika Mittge for technical assistance, Rose Gaudreau and the staff of theUniversity of Oregon Zebrafish Facility for excellent fish husbandry, Poh Kheng Loiand the staffs of the University of Oregon and Oregon State University histologyfacilities for histology services.

COMPETING INTERESTSThe authors declare that they do not have any competing or financial interests.

AUTHOR CONTRIBUTIONSJ.T.N. and K.G. designed experiments. J.T.N. performed experiments. J.T.N., T.S.P.,M.L.K. and K.G. analyzed data. J.T.N., T.S.P., M.L.K. and K.G. wrote the paper.

FUNDINGThis research was supported by the National Institutes of Health (NIH) [grantnumber 1R01DK075667 to K.G.] and a Burroughs Wellcome Fund Investigator inthe Pathogenesis of Infectious Disease Award (to K.G.). The NIH [grant numberHD22486] provided support for the Oregon Zebrafish Facility.

SUPPLEMENTARY MATERIALSupplementary material for this article is available athttp://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.011163/-/DC1

REFERENCESAmieva, M. R. and El-Omar, E. M. (2008). Host-bacterial interactions in Helicobacter

pylori infection. Gastroenterology 134, 306-323.Amieva, M. R., Vogelmann, R., Covacci, A., Tompkins, L. S., Nelson, W. J. and Falkow,

S. (2003). Disruption of the epithelial apical-junctional complex by Helicobacter pyloriCagA. Science 300, 1430-1434.

Asghar, R. J. and Parsonnet, J. (2001). Helicobacter pylori and risk for gastricadenocarcinoma. Semin. Gastrointest. Dis. 12, 203-208.

Barker, N., Huch, M., Kujala, P., van de Wetering, M., Snippert, H. J., van Es, J. H., Sato,T., Stange, D. E., Begthel, H., van den Born, M. et al. (2010). Lgr5(+ve) stem cells driveself-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6,25-36.

Berghmans, S., Murphey, R. D., Wienholds, E., Neuberg, D., Kutok, J. L., Fletcher, C. D.,Morris, J. P., Liu, T. X., Schulte-Merker, S., Kanki, J. P. et al. (2005). tp53 mutantzebrafish develop malignant peripheral nerve sheath tumors. Proc. Natl. Acad. Sci. USA102, 407-412.

Blaser, M. J. and Atherton, J. C. (2004). Helicobacter pylori persistence: biology anddisease. J. Clin. Invest. 113, 321-333.

Bourzac, K. M. and Guillemin, K. (2005). Helicobacter pylori-host cell interactionsmediated by type IV secretion. Cell. Microbiol. 7, 911-919.

Buti, L., Spooner, E., Van der Veen, A. G., Rappuoli, R., Covacci, A. and Ploegh, H. L.(2011). Helicobacter pylori cytotoxin-associated gene A (CagA) subverts the apoptosis-stimulating protein of p53 (ASPP2) tumor suppressor pathway of the host. Proc. Natl.Acad. Sci. USA 108, 9238-9243.

Censini, S., Lange, C., Xiang, Z., Crabtree, J. E., Ghiara, P., Borodovsky, M., Rappuoli,R. and Covacci, A. (1996). cag, a pathogenicity island of Helicobacter pylori, encodestype I-specific and disease-associated virulence factors. Proc. Natl. Acad. Sci. USA 93,14648-14653.

Ceol, C. J., Houvras, Y., Jane-Valbuena, J., Bilodeau, S., Orlando, D. A., Battisti, V.,Fritsch, L., Lin, W. M., Hollmann, T. J., Ferré, F. et al. (2011). The histonemethyltransferase SETDB1 is recurrently amplified in melanoma and accelerates itsonset. Nature 471, 513-517.

Cheesman, S. E., Neal, J. T., Mittge, E., Seredick, B. M. and Guillemin, K. (2011).Epithelial cell proliferation in the developing zebrafish intestine is regulated by the Wntpathway and microbial signaling via Myd88. Proc. Natl. Acad. Sci. USA 108 Suppl. 1,4570-4577.

Churin, Y., Al-Ghoul, L., Kepp, O., Meyer, T. F., Birchmeier, W. and Naumann, M. (2003).Helicobacter pylori CagA protein targets the c-Met receptor and enhances themotogenic response. J. Cell Biol. 161, 249-255.

Crosnier, C., Vargesson, N., Gschmeissner, S., Ariza-McNaughton, L., Morrison, A. andLewis, J. (2005). Delta-Notch signalling controls commitment to a secretory fate in thezebrafish intestine. Development 132, 1093-1104.

El-Etr, S. H., Mueller, A., Tompkins, L. S., Falkow, S. and Merrell, D. S. (2004).Phosphorylation-independent effects of CagA during interaction between Helicobacterpylori and T84 polarized monolayers. J. Infect. Dis. 190, 1516-1523.

El-Omar, E. M., Rabkin, C. S., Gammon, M. D., Vaughan, T. L., Risch, H. A., Schoenberg,J. B., Stanford, J. L., Mayne, S. T., Goedert, J., Blot, W. J. et al. (2003). Increased risk ofnoncardia gastric cancer associated with proinflammatory cytokine genepolymorphisms. Gastroenterology 124, 1193-1201.

Faro, A., Boj, S. F. and Clevers, H. (2009). Fishing for intestinal cancer models: unravelinggastrointestinal homeostasis and tumorigenesis in zebrafish. Zebrafish 6, 361-376.

Fearon, E. R. and Vogelstein, B. (1990). A genetic model for colorectal tumorigenesis.Cell 61, 759-767.

Feng, H., Stachura, D. L., White, R. M., Gutierrez, A., Zhang, L., Sanda, T., Jette, C.A., Testa, J. R., Neuberg, D. S., Langenau, D. M. et al. (2010). T-lymphoblasticlymphoma cells express high levels of BCL2, S1P1, and ICAM1, leading to a blockadeof tumor cell intravasation. Cancer Cell 18, 353-366.

Franco, A. T., Israel, D. A., Washington, M. K., Krishna, U., Fox, J. G., Rogers, A. B.,Neish, A. S., Collier-Hyams, L., Perez-Perez, G. I., Hatakeyama, M. et al. (2005).

Dise

ase

Mod

els &

Mec

hani

sms

D

MM



Activation of beta-catenin by carcinogenic Helicobacter pylori. Proc. Natl. Acad. Sci.USA 102, 10646-10651.

Guillemin, K., Salama, N. R., Tompkins, L. S. and Falkow, S. (2002). Cagpathogenicity island-specific responses of gastric epithelial cells to Helicobacterpylori infection. Proc. Natl. Acad. Sci. USA 99, 15136-15141.

Haramis, A. P., Hurlstone, A., van der Velden, Y., Begthel, H., van den Born, M.,Offerhaus, G. J. and Clevers, H. C. (2006). Adenomatous polyposis coli-deficientzebrafish are susceptible to digestive tract neoplasia. EMBO Rep. 7, 444-449.

He, T. C., Sparks, A. B., Rago, C., Hermeking, H., Zawel, L., da Costa, L. T., Morin, P.J., Vogelstein, B. and Kinzler, K. W. (1998). Identification of c-MYC as a target of theAPC pathway. Science 281, 1509-1512.

Heisenberg, C. P., Houart, C., Take-Uchi, M., Rauch, G. J., Young, N., Coutinho, P.,Masai, I., Caneparo, L., Concha, M. L., Geisler, R. et al. (2001). A mutation in theGsk3-binding domain of zebrafish Masterblind/Axin1 leads to a fate transformationof telencephalon and eyes to diencephalon. Genes Dev. 15, 1427-1434.

Her, G. M., Chiang, C. C. and Wu, J. L. (2004). Zebrafish intestinal fatty acid bindingprotein (I-FABP) gene promoter drives gut-specific expression in stable transgenicfish. Genesis 38, 26-31.

Higashi, H., Tsutsumi, R., Muto, S., Sugiyama, T., Azuma, T., Asaka, M. andHatakeyama, M. (2002). SHP-2 tyrosine phosphatase as an intracellular target ofHelicobacter pylori Cag. Science 295, 683-686.

Higashi, H., Nakaya, A., Tsutsumi, R., Yokoyama, K., Fujii, Y., Ishikawa, S., Higuchi,M., Takahashi, A., Kurashima, Y., Teishikata, Y. et al. (2004). Helicobacter pyloriCagA induces Ras-independent morphogenetic response through SHP-2 recruitmentand activation. J. Biol. Chem. 279, 17205-17216.

Higashijima, S., Okamoto, H., Ueno, N., Hotta, Y. and Eguchi, G. (1997). High-frequency generation of transgenic zebrafish which reliably express GFP in wholemuscles or the whole body by using promoters of zebrafish origin. Dev. Biol. 192,289-299.

Kim, B. M., Buchner, G., Miletich, I., Sharpe, P. T. and Shivdasani, R. A. (2005). Thestomach mesenchymal transcription factor Barx1 specifies gastric epithelial identitythrough inhibition of transient Wnt signaling. Dev. Cell 8, 611-622.

Kwan, K. M., Fujimoto, E., Grabher, C., Mangum, B. D., Hardy, M. E., Campbell, D.S., Parant, J. M., Yost, H. J., Kanki, J. P. and Chien, C. B. (2007). The Tol2kit: amultisite gateway-based construction kit for Tol2 transposon transgenesisconstructs. Dev. Dyn. 236, 3088-3099.

Lee, A., O’Rourke, J., De Ungria, M. C., Robertson, B., Daskalopoulos, G. andDixon, M. F. (1997). A standardized mouse model of Helicobacter pylori infection:introducing the Sydney strain. Gastroenterology 112, 1386-1397.

Mimuro, H., Suzuki, T., Tanaka, J., Asahi, M., Haas, R. and Sasakawa, C. (2002).Grb2 is a key mediator of helicobacter pylori CagA protein activities. Mol. Cell 10,745-755.

Muncan, V., Faro, A., Haramis, A. P., Hurlstone, A. F., Wienholds, E., van Es, J.,Korving, J., Begthel, H., Zivkovic, D. and Clevers, H. (2007). T-cell factor 4 (Tcf7l2)maintains proliferative compartments in zebrafish intestine. EMBO Rep. 8, 966-973.

Murata-Kamiya, N., Kurashima, Y., Teishikata, Y., Yamahashi, Y., Saito, Y., Higashi,H., Aburatani, H., Akiyama, T., Peek, R. M., Jr, Azuma, T. et al. (2007). Helicobacterpylori CagA interacts with E-cadherin and deregulates the beta-catenin signal thatpromotes intestinal transdifferentiation in gastric epithelial cells. Oncogene 26, 4617-4626.

Nagy, T. A., Wroblewski, L. E., Wang, D., Piazuelo, M. B., Delgado, A., Romero-Gallo, J., Noto, J., Israel, D. A., Ogden, S. R., Correa, P. et al. (2011). β-Catenin andp120 mediate PPARδ-dependent proliferation induced by Helicobacter pylori inhuman and rodent epithelia. Gastroenterology 141, 553-564.

Ng, A. N., de Jong-Curtain, T. A., Mawdsley, D. J., White, S. J., Shin, J., Appel, B.,Dong, P. D., Stainier, D. Y. and Heath, J. K. (2005). Formation of the digestivesystem in zebrafish: III. Intestinal epithelium morphogenesis. Dev. Biol. 286, 114-135.

Nobili, S., Bruno, L., Landini, I., Napoli, C., Bechi, P., Tonelli, F., Rubio, C. A., Mini, E.and Nesi, G. (2011). Genomic and genetic alterations influence the progression ofgastric cancer. World J. Gastroenterol. 17, 290-299.

Ohnishi, N., Yuasa, H., Tanaka, S., Sawa, H., Miura, M., Matsui, A., Higashi, H.,Musashi, M., Iwabuchi, K., Suzuki, M. et al. (2008). Transgenic expression ofHelicobacter pylori CagA induces gastrointestinal and hematopoietic neoplasms inmouse. Proc. Natl. Acad. Sci. USA 105, 1003-1008.

Ootani, A., Li, X., Sangiorgi, E., Ho, Q. T., Ueno, H., Toda, S., Sugihara, H., Fujimoto,K., Weissman, I. L., Capecchi, M. R. et al. (2009). Sustained in vitro intestinalepithelial culture within a Wnt-dependent stem cell niche. Nat. Med. 15, 701-706.

Oshima, H., Matsunaga, A., Fujimura, T., Tsukamoto, T., Taketo, M. M. and Oshima,M. (2006). Carcinogenesis in mouse stomach by simultaneous activation of the Wntsignaling and prostaglandin E2 pathway. Gastroenterology 131, 1086-1095.

Peek, R. M., Jr and Blaser, M. J. (2002). Helicobacter pylori and gastrointestinal tractadenocarcinomas. Nat. Rev. Cancer 2, 28-37.

Pinto, D., Gregorieff, A., Begthel, H. and Clevers, H. (2003). Canonical Wnt signalsare essential for homeostasis of the intestinal epithelium. Genes Dev. 17, 1709-1713.

Powell, S. M., Zilz, N., Beazer-Barclay, Y., Bryan, T. M., Hamilton, S. R., Thibodeau,S. N., Vogelstein, B. and Kinzler, K. W. (1992). APC mutations occur early duringcolorectal tumorigenesis. Nature 359, 235-237.

Ranzani, G. N., Luinetti, O., Padovan, L. S., Calistri, D., Renault, B., Burrel, M.,Amadori, D., Fiocca, R. and Solcia, E. (1995). p53 gene mutations and proteinnuclear accumulation are early events in intestinal type gastric cancer but lateevents in diffuse type. Cancer Epidemiol. Biomarkers Prev. 4, 223-231.

Ruijter, J. M., Ramakers, C., Hoogaars, W. M., Karlen, Y., Bakker, O., van den Hoff,M. J. and Moorman, A. F. (2009). Amplification efficiency: linking baseline and biasin the analysis of quantitative PCR data. Nucleic Acids Res. 37, e45.

Sato, N., Meijer, L., Skaltsounis, L., Greengard, P. and Brivanlou, A. H. (2004).Maintenance of pluripotency in human and mouse embryonic stem cells throughactivation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat. Med.10, 55-63.

Sato, T., van Es, J. H., Snippert, H. J., Stange, D. E., Vries, R. G., van den Born, M.,Barker, N., Shroyer, N. F., van de Wetering, M. and Clevers, H. (2011). Paneth cellsconstitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469, 415-418.

Segal, E. D., Cha, J., Lo, J., Falkow, S. and Tompkins, L. S. (1999). Altered states:involvement of phosphorylated CagA in the induction of host cellular growthchanges by Helicobacter pylori. Proc. Natl. Acad. Sci. USA 96, 14559-14564.

Selbach, M., Moese, S., Hauck, C. R., Meyer, T. F. and Backert, S. (2002). Src is thekinase of the Helicobacter pylori CagA protein in vitro and in vivo. J. Biol. Chem. 277,6775-6778.

Shibata, A., Parsonnet, J., Longacre, T. A., Garcia, M. I., Puligandla, B., Davis, R. E.,Vogelman, J. H., Orentreich, N. and Habel, L. A. (2002). CagA status ofHelicobacter pylori infection and p53 gene mutations in gastric adenocarcinoma.Carcinogenesis 23, 419-424.

Solnick, J. V., Canfield, D. R., Yang, S. and Parsonnet, J. (1999). Rhesus monkey(Macaca mulatta) model of Helicobacter pylori: noninvasive detection and derivationof specific-pathogen-free monkeys. Lab. Anim. Sci. 49, 197-201.

Stein, M., Bagnoli, F., Halenbeck, R., Rappuoli, R., Fantl, W. J. and Covacci, A.(2002). c-Src/Lyn kinases activate Helicobacter pylori CagA through tyrosinephosphorylation of the EPIYA motifs. Mol. Microbiol. 43, 971-980.

Storer, N. Y. and Zon, L. I. (2010). Zebrafish models of p53 functions. Cold Spring Harb.Perspect. Biol. 2, a001123.

Suzuki, M., Mimuro, H., Suzuki, T., Park, M., Yamamoto, T. and Sasakawa, C. (2005).Interaction of CagA with Crk plays an important role in Helicobacter pylori-inducedloss of gastric epithelial cell adhesion. J. Exp. Med. 202, 1235-1247.

Suzuki, M., Mimuro, H., Kiga, K., Fukumatsu, M., Ishijima, N., Morikawa, H., Nagai,S., Koyasu, S., Gilman, R. H., Kersulyte, D. et al. (2009). Helicobacter pylori CagAphosphorylation-independent function in epithelial proliferation and inflammation.Cell Host Microbe 5, 23-34.

Tetsu, O. and McCormick, F. (1999). Beta-catenin regulates expression of cyclin D1 incolon carcinoma cells. Nature 398, 422-426.

Tissier-Seta, J. P., Mucchielli, M. L., Mark, M., Mattei, M. G., Goridis, C. and Brunet,J. F. (1995). Barx1, a new mouse homeodomain transcription factor expressed incranio-facial ectomesenchyme and the stomach. Mech. Dev. 51, 3-15.

Tsukamoto, T., Mizoshita, T., Mihara, M., Tanaka, H., Takenaka, Y., Yamamura, Y.,Nakamura, S., Ushijima, T. and Tatematsu, M. (2005). Sox2 expression in humanstomach adenocarcinomas with gastric and gastric-and-intestinal-mixedphenotypes. Histopathology 46, 649-658.

Tsutsumi, R., Higashi, H., Higuchi, M., Okada, M. and Hatakeyama, M. (2003).Attenuation of Helicobacter pylori CagA x SHP-2 signaling by interaction betweenCagA and C-terminal Src kinase. J. Biol. Chem. 278, 3664-3670.

van de Water, S., van de Wetering, M., Joore, J., Esseling, J., Bink, R., Clevers, H.and Zivkovic, D. (2001). Ectopic Wnt signal determines the eyeless phenotype ofzebrafish masterblind mutant. Development 128, 3877-3888.

Westerfield, M. (2007). The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish(Danio rerio). Eugene, OR: University of Oregon Press.

White, R. M., Cech, J., Ratanasirintrawoot, S., Lin, C. Y., Rahl, P. B., Burke, C. J.,Langdon, E., Tomlinson, M. L., Mosher, J., Kaufman, C. et al. (2011). DHODHmodulates transcriptional elongation in the neural crest and melanoma. Nature 471,518-522.

Wirth, H. P., Beins, M. H., Yang, M., Tham, K. T. and Blaser, M. J. (1998).Experimental infection of Mongolian gerbils with wild-type and mutant Helicobacterpylori strains. Infect. Immun. 66, 4856-4866.

Yan, D., Wiesmann, M., Rohan, M., Chan, V., Jefferson, A. B., Guo, L., Sakamoto, D.,Caothien, R. H., Fuller, J. H., Reinhard, C. et al. (2001). Elevated expression of axin2and hnkd mRNA provides evidence that Wnt/beta-catenin signaling is activated inhuman colon tumors. Proc. Natl. Acad. Sci. USA 98, 14973-14978.

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h. pylori virulence factor caga increases intestinal cell ... · gastritis, peptic ulcer disease...

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